Download Johnson, H. N. Purification of

Johnson, H. N. Purification of
Neurospora
This communication will describe on efficient method for the purification of
the rmaller B-galactoridare of Neurorpora (pH 4.2 enzyme). Wild type Neuror-
R-golactoridare.
porn
cras~o
(74-OR-A) wm used
for the isolation of the enzyme.
Large quontitier
fiyxwere prepared by incubating conidia in ten liten of Vogel’s minimal
roltr plus 0 . 7 % L-arobinme and 0 . 3 % sucrose. Tne culture wm aerated by bubbl’ an g and inwbated f o r 9 6 hours ot 25’C. The
culture was harvested by straining through cheese-cloth, washing with cold distilled water, and compressing the myceliol mat by
vacuum filtration. Cells were disrupted for protein extraction in o Sorvall Ovni-Mixer with 0.01 M patorrium phmphote buffer,
Bioronik
at moxipH 7.5 (&ffer A) at a ratio of 6 ml of buffer/g of mycelia.
Th’ II was followed by sonication with o Bronwill
mum probe intensity for 30 ret per 100 ml of homogenate. All procedures were carried
rhoken for 2 hrr and then centrifuged at 27,000 x g for 20 min.
Table 1. Summary
out at 4’C.
The homogenate was gently
The supernotont w e wed as the crude extract.
of plrificotion.
Table 2. Amino acid composition of the
step
Volume
Protein
ml
mg
Total
activity
Specific
e n z y m e units
Recovery
activity
pH
4.2 R-goloctoridore
63
I . C r u d e extract
2. 75% AmSO
200
3,745
20,255
100
1,075
15,333
Amino
100
5.4
14.1
75
acid
80.77
81
Histidine
14.48
0
I5
0
EX.58
43. I8
85
43
202.06
140.84
202
Glyche
26.04
258.57
26
259
Alanine
Cystine
92.33
____
92
__
Valine
Methionine
22.22
I .27
22
I
100
178
15,333
86. I
76
4. After
II0
38
15,000
392
74
6.6
13,775
2078
60
Swine
Glutomic
9,925
33,250
49
Proline
5
ppt
.
753bAmS04
after dialysis
6. CM
eluant
5
The crude extract is
fate a+ pH 7.5 and
2.
0.298
taken to 33% saturation with rlmmonium
stirred for 20 min.
After centrifugation
at
Arginine
Arpartic acid
Threonine
sul27,000
Residuer/MW of 96,000
Neorert integer
Found
Lysine
PP’
3. pH 4.Oroluble
dialysis
of Neurorporo 74A.
%
acid
141
x g for 20 min., the rupernotant is token to 75% soturotion with ommonium
sulfate at pH 5.0 and stirred for 4 hn.
The precipitate is collected by centrifugotion at 27,000 x g and dissolved in l/IO the orig-
lroleucine
14.48
I5
26.04
26
inal volume of Buffer A. This solution is made pH 4.0 by the addition
of I M citric acid and allwed to stand in the cold for 8 hn.
The precipitate is centrifuged out and the supernatant is dialyzed against dirtilled water for 6 hn.
The dialyzed rupernatant from centrifugation is
Leucine
Tyrosine
Renylolanine
toto I
13.21
7.49
13
8
taken to 75% saturation with ammonium sulfate (I+ pH 5.0 and allowed
to stand for IO-12 hrr.
The precipitate is collected and dissolved in l/l00
phote-citrate,
pH
1,029
the original volume of Euffer B (0.008 M Sodium phcs-
4.2) with 0.01 M 2-mercaptoethanol.
This solution is then chromatographed on a CM Sephodex column equilibrated with &Iffer B plus mercaptcethonol. The enzyme is eluted with a NoCl gradient (0.008-1.0 M in Buffer B). The peak
fractions contain an electrophoretically
pure enzyme preparation. As seen in Table I, the overall purification is some 6000-fold
with
a 49% recovery.
A sample of the purified enzyme
I208
automatic
was hydrolyzed in b N HCI and its amino acid composition war determined with
amino acid analyzer. The amino acid composition is
shown
in Table 2.
a Beckman
T h e number of residues ,.,a~ bored on ~1
molecular weight of 96,000. The composition was determined on the botir of three individually purified sampler.
The abence
of orginine in this enzyme may be an important point in future work with this enzyme, especially with respect to peptide mapping.
This work supported in part by the NIH Training Grant in Genetics (TOI-GM01316) to Florida State University - - - Genetics
Laboratories, Deportment of Biological Science, Florida State University, Tallahassee, Florida 32306.
Johnson, H. N. Identification of a third farm
of D-goloctoridare
by sectioning ocrylamide
previously described B-goloctoridorer
pH optimum at 4.5.
The rtondord
gels.
with pH
Strickland and Shields (1967 Neurorporo Nwrl. 12: 15) described o
method for rpecifically staining enzymes and matching them to proteins stained
on the rome ocrylamide gel. This communication describer a more satisfactory
methcd for identifying the R-golactosidoses in Neurcsporo. In addition to the
optima at 7.5 and 4.2, o third form has been identified
7.5% gels were used and after electrophorerir
b this
methcd which has
were frozen on dry ice or by immersion in liquid nitrogen.
D
The
gels were then split lengthwise with D razor blade and one half wets stained with amide block.
The other half WOI then sectioned
into l-mm sections by a gel slicer. The slices were individually mroyed wing 0-nitro
phenyl-R-D-gclloctopyranoside.
The enzyme activity for each farm of the enzyme uruolly WOI localized in 2 or 3 slices.
front marker, Rf’r for the enzymes were fairly reproducible.
Lking
tracking dye (brom
phenol blue)
os D
W i l d ‘ype Neurosporo crosso
(74-OR-A) woz grown on 196 lactose for 5 days and the mycelio were filtered out on o &hoer
The mycelio were homefunnel.
The mycelio were extracted with 10 ml of 0.01 M No phosphate, pH 7.5, per g of wet weight.
genized
in on Omni-Mixer, sonicoted, and then stirred for 2 hrs ot 4°C.
noton+ war used os crude extract.
for electrophoresis
contained
The growth medium after filtration
After centrifugotion
at 20,000 x g for 20 min, the super-
wm concentrated by dialysis
ogointt dry sucro~c.
Sampler
z 0.25 mg protein.
Crude extract gave reoctiom with ONPG ot three distinct sites on the gel,
The 7.5 enzyme hod on Rf of 0.046, the 4.2 en-
zyme hod on Rf of 0.250, ond o third form of the enzyme hod on Rf of 0. 150.
When the growth medium was electrophoresed, octivity oppeored
ot either one or two sites, depending upon the age of the culture. Medium from o young culture shaved only the
I e medium from on old culture contained both the 4.2 and 4.5 forms with o predominance
new form of the enzyme (pH 4.5) w h.1
of the former.
by the NIH
Biological
Between these two extremes there were gradations in the proportions of the two forms. This work supported in port
Training Grant
Science,
in Genetics (TOI-GMOl316)
Florida
State
University,
Morgan, D. H. The msay of orginose.
Frozen myceliol
to Florida State University. - - - Genetics Laboratories, Department of
Tollohossee,
Florida
32306.
Many methods of orginore ossoy in voriws organisms hove been published. The
foilwing procedure has been found to tiork well with crude extracts of Neuraporo.
pods are ground in o chilled mortar with gloss powder and 5-10 times their weight of
dithioerythritol (0.002 M).
sodium hydroxide buffer containing mongonese
chloride (0.005 M) and
superfluous in undiolyned extracts but has been found to stobilire
the enzyme during dialysis.
A reaction mixture cdnsists simply of 0.2 m! of enzyme ond 0.3 ml of 0.3 M orginine.
9.5 and
-SH
reagent
The orginine solution is
is
possibly
adjusted to
pH
&out 9.2, the orginine itself prov’oviding
adequate buffering. Incubation is ot 37”C, the enzyme beThe reaction is stopped with 4.5 ml of 2% TCA,
ing po-incubated
ot this temperature for IO-15 min before arginine addition.
zero-time blanks being stopped before orginine addition.
Ornithine
estimation (see below ) is carried out on 0.5 ml samples of
the stopped reaction mix.
(Wh en it is desired to use lower sutstrote concentrations ond therefore to detect lower levels of ornithin=,
final
pH 7.0 0.025 M moleic acid/
An
PH in the mix is
to stay in the linear region dilution with TCA is reduced or avoided oltcgether
used for the ornithine
The estimation
estimation
by stopping with the
acid ninhydrin reagent
estimation.)
of ornithine in assaying for
orginare or
acetyl-ornithine/glutclmate
tronsacetylose.
used by Vogel and Banner ( 1956 J. Riol. them. 218:97) for the ossoy of ocetylornithinose
The method of ornithine
is also applicable
to the
array of arginase and ocetylornithine/glutomo+e
tronsacetylose. It is quicker than the commonly-used method of Chinord (1952
J. Biol. Chem. 199: 91 ) ond the ninhydrin mix used (mode up in 0.4 hl citric acid and methyl cellosolve) is pleosonter to deol
with than that of Chinord (6 M phosphoric acid ond glacial acetic acid).
The optimum boiling time for arginose samples is 25
min.
Both glutornate and orginine give rise to a deep blue color after the final addition of NoOH.
This persish until the samples
ore subjected to vigorous Vortex mixing (30-60 set ) when it disoppeors, revealing the stable golden-brown color which is read
ot 470 mp.
It is necessary to read ogoinst no-ornithine blanks containing appropriate quantities of orginine or glutamate, both of
which give appreciable blank values. Sensitivity is obcut three-fold lower than with Chinard - 0.4 pmoles of ornithine per OD
unit ot 470 mp in the presence of 9 pmoles orginine per sample. The reaction is lineor ot least up to 0.5 pmoles ornithine per
SOfllpl@.
This work was supported by the Gceney
Fund, Colifornio Institute of Technology, The hospitality and encouragement
N. H. Horowitz was greatly appreciated. - - - John Inner Institute, Colney Lone, Norwich NOR 7OF. England.
Flovell,
The following ossoyr hove been used for Neurosporo
R. Comments on arsoy methods.
Finchom
(1968 J. Eocteriol.
os reported by Flovell
95: 1063). Any modifications
noted after the relevant osroy.
the paper mentioned above.
The original
of
and
used since then ore
references to the ossoys
ore given in
lsocitrote Iyore, E.C. 4. I .3. I.; but with I5 pmoles
of DL-isocitrote.
Molote synthase, E.C. 4. I .3.2.; but with 0.04 pmoles of Acetyl C oA.
Phcsphoenolpyruvote
corboxykinase, E.C 4. I. 1.32.; but with 9 pmoles phosphoenolpyruvote.
Citrotesynthore,
E.C. 4.1.3.7.;
butwith
0.04 pmoles of Acetyl CoA.
Acetyl CoA synthetose E.C. 6.2. I. I.
Molote
dehydrogenose, E.C.
I. I.
1.37.
N A D P - l i n k e d lsocitrote dehydrogenose, E.C. I. I. 1 . 4 2 .
NAD-linked lsocitrote dehydrcgenase, E.C. I. I. 1.41.; but with 8 mM DL-isoctote and I
Fumorote
hydrotose, E.C. 4.2.
The following assays have also been
used without modification of the method cited in the reference following each enzyme:
Succinote dehydrogenase, E . C . 1.3.99. I. ( K ing
A T P citrate lyase, E.C. 4 . 1.3.6. ( A t k i n s o n
-
mM NAD.
I. 2.
- - Department of Biological Sciences,
Sfonford
1963 J .
Biol.
C h e m . 238:4032)
Biochem. Sot. Symp. 27:31 ).
University, Stanford, Colifornio
94305.